The duckweeds are the sole members of the monocotyledonous family Lemnaceae. The four genera and 34 species are all small, free-floating, fresh-water plants whose geographical range spans the entire globe (Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The Family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich). Although the most morphologically reduced plants known, most duckweed species have all the tissues and organs of much larger plants, including roots, stems, flowers, seeds and fronds. Duckweed species have been studied extensively and a substantial literature exists detailing their ecology, systematics, life-cycle, metabolism, disease and pest susceptibility, their reproductive biology, genetic structure, and cell biology. (Hillman (1961) Bot. Review 27: 221; Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The Family of Lemnaceae—A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich).
The growth habit of the duckweeds is ideal for microbial culturing methods. The plant rapidly proliferates through vegetative budding of new fronds, in a macroscopic manner analogous to asexual propagation in yeast. This proliferation occurs by vegetative budding from meristematic cells. The meristematic region is small and is found on the ventral surface of the frond. Meristematic cells lie in two pockets, one on each side of the frond midvein. The small midvein region is also the site from which the root originates and the stem arises that connects each frond to its mother frond. The meristematic pocket is protected by a tissue flap. Fronds bud alternately from these pockets. Doubling times vary by species and are as short as 20-24 hours (Landolt (1957) Ber. Schweiz. Bot. Ges. 67: 271; Chang et al. (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman et al. (1970) Z. Pflanzenphysiol. 62: 316).
Duckweed plant or duckweed nodule cultures can be efficiently transformed by any one of a number of methods including Agrobacterium-mediated gene transfer, ballistic bombardment, or electroporation. Stable duckweed transformants can be isolated by transforming the duckweed cells with both the nucleotide sequence of interest and a gene which confers resistance to a selection agent, followed by culturing the transformed cells in a medium containing the selection agent. See U.S. Pat. No. 6,040,498 and U.S. Provisional Patent Application No. 60/221,705; herein incorporated by reference in their entirety.
Recent advances in plant genome sequencing have provided a wealth of information regarding the sequence of genes encoding novel polypeptides and the associated nucleotide sequences that may serve to regulate the expression of these coding sequences. However, these sequence data provide no information regarding the precise role of these nucleotide sequences in physiologically-important processes.
To determine or confirm the function of novel genes, it is generally necessary to express these genes in vivo. When screening nucleotide sequences to determine their activities in plants, the highest predictive success comes from tests for the activity of the sequence in a whole plant. However, greenhouse testing of intact plants grown in soil is time- and space-consuming. Large amounts of labor are also required to prepare, care for, and score these screens, and these steps are generally not amenable to automation.
A method for screening for nucleotide sequences in a system that combines the predictive success of whole plant screening with the reduced size and cost of high-throughput screening is desirable. Accordingly, the present invention provides methods for conducting high throughput screens in a duckweed system.